Glioblastomas (GBMs) are the most common and aggressive primary brain tumors in adults, remaining as one of the deadliest forms of cancer. These tumors are highly invasive, which makes their complete resection impossible and contributes to their recurrence and lethality. Changes in cell polarity are critical for tumor invasion in response to signals from the tumor microenvironment; however, these mechanisms have been largely overlooked in GBMs. Our goal in this project is to investigate a novel cell polarity target that may underlie the ability of GBM cells to invade in response to specific signals produced by neural cells. The proteins of the DLG family contribute to normal cell polarity by regulating the transport of signaling molecules between the apical and basolateral domains of normal cells. These proteins are downregulated in epithelial tumors and are thought to act as tumor suppressors. However, our preliminary work has revealed that the protein DLG5 has a striking upregulation in GBMs and contributes to tumor invasion and activation of the Sonic Hedgehog (SHH) pathway in GBM stem cells. Our central hypothesis is that DLG5 plays a unique tumor-promoting role in GBMs and regulates the ability of invasive GBM cells to respond to guidance signals (i.e., SHH ligand) originated from neural cells. To test this hypothesis, our first Aim is to determine the functions of DLG5 on GBM growth, dispersion, and tropism towards neural cells. Using an inducible-shRNA strategy we will study how the loss of DLG5 affects tumor growth, invasion, and interaction of GBM cells with oligodendrocyte precursors that are a major source of SHH ligand in adult brain. Our second aim is to determine if DLG5 specifically regulates pro- invasive SHH signaling in GBM stem cells. We will study the molecular association of DLG5 with components of the SHH pathway and will analyze how DLG5 knockdown affects this pathway in GBM cells in vitro and in vivo. To our knowledge, this is the first investigation of the DLG family of cell polarity proteins in GBMs, and the first study to propose that DLG5 may have a tumor-promoting function and is required for interactions between brain tumor cells and their unique neural microenvironment. Successful completion of the project will help understand the communication that occurs between glioma cells and neural cells during tumor dispersion, allowing us to formulate strategies to disrupt those interactions. Our studies will help disable tumor cells that remain in the brain after surgery, which could potentiate therapies to prevent recurrence and achieve long-term survival in patients with malignant gliomas.